U.S. patent application number 10/704229 was filed with the patent office on 2004-09-02 for anti-tumor antigen against htlv-i tumor or antigen epitope thereof.
Invention is credited to Hanabuchi, Shino, Kannagi, Mari, Ohashi, Takashi.
Application Number | 20040171090 10/704229 |
Document ID | / |
Family ID | 18984621 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040171090 |
Kind Code |
A1 |
Hanabuchi, Shino ; et
al. |
September 2, 2004 |
Anti-tumor antigen against HTLV-I tumor or antigen epitope
thereof
Abstract
The present invention provides a immunogenic composition for
inducing an immune response containing a CTL recognition antigen or
an antigen epitope thereof, which can be obtained by screening a
CTL recognition antigen or an antigen epitope thereof having an
anti-tumor effect against HTLV-I tumors such as ATL, or a DNA that
encodes them as an active ingredient and the like. Dominant epitope
GAFLTNVPY was identified by the following steps: splenic T cells
derived from immunocompetent rats immunized with HTLV-I-infected
cell lines were stimulated with formalin-fixed HTLV-I-infected cell
lines; HTLV-I-specific CTL cell lines were established; cytotoxic
activities of the above-mentioned HTLV-I-specific CTL cell lines
against target cells G14 sensitized with synthetic peptides which
are candidate for an epitope were measured. By immunizing
immunocompetent rats with said epitope synthetic peptide and an
adjuvant, then inducing tumor antigen epitope-specific CTLs, the
proliferation of HTLV-I-infected tumor cells in vivo can be
suppressed.
Inventors: |
Hanabuchi, Shino; (Houston,
TX) ; Ohashi, Takashi; (Tokyo, JP) ; Kannagi,
Mari; (Tokyo, JP) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
18984621 |
Appl. No.: |
10/704229 |
Filed: |
November 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10704229 |
Nov 7, 2003 |
|
|
|
PCT/JP02/04406 |
May 2, 2002 |
|
|
|
Current U.S.
Class: |
435/7.23 ;
530/350 |
Current CPC
Class: |
A61P 37/04 20180101;
A61K 2039/515 20130101; A61K 2039/53 20130101; A61K 39/00 20130101;
A61P 35/02 20180101; A61K 39/0011 20130101; G01N 2333/15 20130101;
A61P 35/00 20180101; A61K 2039/57 20130101; G01N 33/6878 20130101;
C07K 14/005 20130101; G01N 33/505 20130101; A61K 49/0008 20130101;
A61K 2035/124 20130101; C12N 2740/14022 20130101 |
Class at
Publication: |
435/007.23 ;
530/350 |
International
Class: |
G01N 033/574; C07K
014/705 |
Foreign Application Data
Date |
Code |
Application Number |
May 8, 2001 |
JP |
2001-137526 |
Claims
We claim:
1. A method of screening for a cytotoxic T-lymphocyte (CTL)
recognition antigen or an antigen epitope thereof that can induce
CTLs having an anti-tumor effect against HTLV-I tumors, wherein:
(a) CTLs induced by a test substance are administered to a
non-human animal model of HTLV-I associated disease, and the change
of tumors in said non-human animal is measured and assessed; or (b)
CTLs induced by a test substance are contacted with HTLV-I infected
tumor cell lines and the cytotoxic activity of said CTLs is
measured and assessed; or (c) target cells sensitized with a test
substance or target cells that express a test substance are
contacted with HTLV-I specific CTL cell lines, and the cytotoxic
activity of said HTLV-I specific CTL cell lines is measured and
assessed.
2. The method of claim 1, wherein the HTLV-I associated disease is
adult T-cell leukemia.
3. The method of claim 1, wherein the non-human animal is a
rat.
4. A CTL recognition antigen or an antigen epitope thereof that can
induce CTLs having an anti-tumor effect against HTLV-I tumors,
which is obtained by the screening method of claim 1.
5. The antigen epitope of claim 4, wherein the antigen epitope is a
peptide comprising an amino acid sequence of SEQ ID NO:2.
6. A method of screening for an adjuvant that enhances an
anti-tumor effect against HTLV-I tumors, wherein: (a) CTLs induced
by using the CTL recognition antigen or antigen epitope of claim 4
and a test adjuvant are administered to a non-human animal model of
HTLV-I associated disease and the change or tumors in said
non-human animals is measured and assessed; or (b) CTLs induced by
using the CTL recognition antigen or antigen epitope of claim 4 and
a test adjuvant are contacted with HTLV-I infected tumor cell
lines, and the cytotoxic activity of said CTLs is measured and
assessed.
7. The method of claim 6, wherein the HTLV-I associated disease is
adult T-cell leukemia.
8. The method of claim 6, wherein the non-human animal is a
rat.
9. An immunogenic composition for inducing an immune response
containing a CTL recognition antigen or an antigen epitope thereof
which can induce CTLs having an anti-tumor effect against HTLV-I
tumors and which can be obtained by the method of claim 1.
10. An immunogenic composition for inducing an immune response
containing a nucleic acid that encodes a CTL recognition antigen or
an antigen epitope thereof which can induce CTLs having an
anti-tumor effect against HTLV-I tumors obtained by the method of
claim 1.
11. The immunogenic composition of claim 9, wherein the CTL
recognition antigen comprises the amino acid sequence of SEQ ID
NO:1.
12. The immunogenic composition of claim 10, wherein the CTL
recognition antigen comprises the amino acid sequence of SEQ ID
NO:1.
13. The immunogenic composition of claim 11, wherein at least one
amino acid is deleted, substituted, or added.
14. The immunogenic composition of claim 12, wherein at least one
amino acid is deleted, substituted, or added.
15. The immunogenic composition of claim 9, wherein the CTL
recognition antigen is a peptide comprising the amino acid sequence
of SEQ ID NO:2.
16 The immunogenic composition of claim 10, wherein the CTL
recognition antigen is a peptide comprising the amino acid sequence
of SEQ ID NO:2.
17. The immunogenic composition of claim 15, wherein at least one
amino acid is deleted, substituted, or added.
18. The immunogenic composition of claim 16, wherein at least one
amino acid is deleted, substituted, or added.
19. The immunogenic composition of claim 9, further comprising an
adjuvant that enhances an anti-tumor effect against HTLV-I.
20. The immunogenic composition of claim 10, further comprising an
adjuvant that enhances an anti-tumor effect against HTLV-I.
21. The immunogenic composition for inducing an immune response
containing a CTL recognition antigen or an antigen epitope thereof
which can induce CTLs having an anti-tumor effect against HTLV-I
tumors and which can be obtained by the method of claim 1, further
comprising an adjuvant that enhances an anti-tumor effect against
HTLV-I, wherein the adjuvant is obtained by the method of screening
for an adjuvant that enhances an anti-tumor effect against HTLV-I
tumors, wherein: (a) CTLs induced by using the CTL recognition
antigen or antigen epitope that can induce CTLs having an
anti-tumor effect against HTLV-I tumors, which is obtained by the
screening method of claim 1 and a test adjuvant are administered to
a non-human animal model of HTLV-I associated disease and the
change or tumors in said non-human animals is measured and
assessed; or (b) CTLs induced by using the CTL recognition antigen
or antigen epitope that can induce CTLs having an anti-tumor effect
against HTLV-I tumors, which is obtained by the screening method of
claim 1 and a test adjuvant are contacted with HTLV-I infected
tumor cell lines, and the cytotoxic activity of said CTLs is
measured and assessed.
22. The immunogenic composition for inducing an immune response
containing a nucleic acid that encodes a CTL recognition antigen or
an antigen epitope thereof which can induce CTLs having an
anti-tumor effect against HTLV-I tumors obtained by the method of
claim 1, further comprising an adjuvant that enhances an anti-tumor
effect against HTLV-I, wherein the adjuvant is obtained by the
method of screening for an adjuvant that enhances an anti-tumor
effect against HTLV-I tumors, wherein: (a) CTLs induced by using
the CTL recognition antigen or antigen epitope that can induce CTLs
having an anti-tumor effect against HTLV-I tumors, which is
obtained by the screening method of claim 1 and a test adjuvant are
administered to a non-human animal model of HTLV-I associated
disease and the change or tumors in said non-human animals is
measured and assessed; or (b) CTLs induced by using the CTL
recognition antigen or antigen epitope that can induce CTLs having
an anti-tumor effect against HTLV-I tumors, which is obtained by
the screening method of claim 1 and a test adjuvant are contacted
with HTLV-I infected tumor cell lines, and the cytotoxic activity
of said CTLs is measured and assessed.
23. The immunogenic composition of claim 19, wherein the adjuvant
is ISS-ODN.
24. The immunogenic composition of claim 20, wherein the adjuvant
is ISS-ODN
25. An HTLV-I recognition CTL induced by the immunogenic
composition of claim 9.
26. An HTLV-I recognition CTL induced by the immunogenic
composition of claim 10.
27. A pharmaceutical composition comprising the HTLV-I recognition
CTL of claim 25.
28. A pharmaceutical composition comprising the HTLV-I recognition
CTL of claim 25.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application PCT/JP02/04406 filed on May 2, 2002 and published as WO
02/090981 A1 on Nov. 14, 2002, which application claims priority
from Japanese Application No. 2001-137526 filed May 8, 2001.
[0002] Each of the foregoing applications, and each document cited
or referenced in each of the foregoing applications, including
during the prosecution of each of the foregoing applications and
("application cited documents"), and any manufacturer 's
instructions or catalogues for any products cited or mentioned in
each of the foregoing applications and articles and in any of the
application cited documents, are hereby incorporated herein by
reference. Furthermore, all documents cited in this text, and all
documents cited or referenced in documents cited in this text, and
any manufacturer's instructions or catalogues for any products
cited or mentioned in this text or in any document hereby
incorporated into this text, are hereby incorporated herein by
reference. Documents incorporated by reference into this text or
any teachings therein may be used in the practice of this
invention. Documents incorporated by reference into this text are
not admitted to be prior art.
[0003] It is noted that in this disclosure and particularly in the
claims, terms such as "comprises", "comprised", "comprising" and
the like can have the meaning attributed to it in U.S. patent law;
e.g., they can mean "includes", "included", "including", and the
like; and that terms such as "consisting essentially of" and
"consists essentially of" have the meaning ascribed to them in U.S.
patent law, e.g., they allow for elements not explicitly recited,
but exclude elements that are found in the prior art or that affect
a basic or novel characteristic of the invention.
FIELD OF THE INVENTION
[0004] The present invention relates to a screening method for a
CTL recognition antigen or an antigen epitope thereof which can
induce cytotoxic T lymphocytes (CTLs) having an anti-tumor effect
against human T cell leukemia virus type I (HTLV-I) tumors such as
adult T cell leukemia (ATL), etc.; a screening method for an
adjuvant that enhances the inducing activity of CTLs having an
anti-tumor effect against HTLV-I tumors by a CTL recognition
antigen or an antigen epitope thereof or an antigen epitope peptide
thereof which can induce CTLs having an anti-tumor effect against
HTLV-I tumors; an immunogenic composition and the like for inducing
an immune response containing said CTL recognition antigen or
antigen epitope thereof, or DNA thereof as an active
ingredient.
BACKGROUND OF THE INVENTION
[0005] HTLV-I is involved in the pathogenesis of ATL,
HTLV-I-associated myelopathy/tropical spastic paraparesis
(HAM/TSP), as well as other inflammatory diseases (Blood 50,
481492, 1977; Proc. Natl. Acad. Sci. USA 77, 7415-7419, 1980; Proc.
Natl. Acad. Sci. USA 78, 6476-6480, 1981; Lancet. 2, 407410, 1985;
Lancet. 1, 1031-1032, 1986). HTLV-I is considered to have a
specific sequence called pX between its env and 3'LTR, wherein this
pX region cannot be seen in carcinogenic retroviruses of other
animals, and its production called Tax protein is considered to
play an important role for various pathogenesis of HTLV-I. It is
also known that said HTLV-I Tax is a virus control protein and can
immortalize rat and human cells in vitro (Proc. Natl. Acad. Sci.
USA 87, 1071-1075, 1990; Blood 86, 4243-4249, 1995; J. Virol. 66,
4570-4575, 1992). These findings strongly indicate that HTLV-I Tax
is involved in the mechanisms of HTLV-I-induced leukemogenesis.
However, the mechanisms of pathogenesis of ATL in vivo are still
unclear.
[0006] A number of studies have revealed that the levels of host
cellular immunity against HTLV-I in ATL patients differ from those
in HAM/TSP patients, and that HTLV-I-specific CTLs can be found in
HAM/TSP patients and asymptomatic HTLV-I carriers but CTLS are only
rarely found in ATL patients (J. Immunol. 130, 2942-2946, 1983; J.
Exp. Med. 158, 994-999, 1983; J. Immunol. 133, 1037-1041, 1984;
Nature 348, 245-248, 1990; Virology 188, 628-636, 1992; Int. J.
Cancer 54, 582-588, 1993). Based on these observations, it is
considered that host cellular immunity may influence pathogenesis
of HTLV-I-associated diseases.
[0007] In general, the above-mentioned CTLs play an important role
not only in viral clearance, but also in tumor eradication. It is
reported by the present inventors and other groups that
HTLV-I-specific CD8.sup.+ CTLs in HTLV-I carriers recognize HTLV-I
Tax (Nature 348, 245-248, 1990; Int. Immunol. 3, 761-767, 1991),
and lyse ATL cells in vitro (J. Immunol. 133, 1037-1041, 1984; Int.
J. Cancer 54, 582-588, 1993). These observations indicate that
HTLV-I-specific CTLs may be an important effector of host
immunosurveillance against HTLV-I-induced tumor proliferation.
However, it is still unclear whether the above-mentioned role of
CTLs is against HTLV-I tumors in vivo or merely a consequence of
infection.
[0008] To clarify the effect of CTLs on HTLV-I leukemogenesis in
vivo, the present inventors developed two experimental rat model
systems of ATL-like diseases (J. Virol. 73, 6031-6040, 1999; J.
Virol. 74, 428-435, 2000; Japanese Patent Application
No.H10-315174). One is a model of T cell lymphomas in athymic rats
following inoculation of HTLV-I-immortalized rat cell lines (J.
Virol. 73, 6031-6040, 1999). In this model, adoptive transfer of
immune T cells protected rats from fatal lymphomas. In the other
model, the development of T cell lymphomas was induced by treatment
with anti-CD80 and -CD86 monoclonal antibodies which block
costimulatory signals for T cell activation in immunocompetent rats
(J. Virol. 74, 428-435, 2000). These findings strongly indicate
that the role of a host T cell immune response as to prevent the
proliferation of HTLV-I tumors in vivo.
[0009] The observations made in studies of CTLs in human
HTLV-I-infected patients and the above-mentioned rat models
indicate that augmentation of HTLV-I-specific CTLs in pre-ATL
patients might protect them from pathogenesis of ATL. Since the
recovery rate of ATL is extremely low among lymphoproliferative
disorders because of its resistance to chemotherapy, the
development of therapies by immunological approach at an early
stage of diseases have been expected. In order to induce an
effective anti-tumor immune response, the tumor antigen must be
precisely clarified and a strong immunogen recognized by cellular
immunity must be administered to the host. In particular, to induce
a CD8.sup.+ CTL response which is one of the major populations that
recognize tumors, presentation of an appropriate peptide processed
by MHC class I as well as class II antigens of antigen-presenting
cells is essential (Nature 343, 692-696; 1989). Because of the
preference of the pathway of antigen presentation by MHC class I, a
variety of viral vectors have been proposed for delivery and
expression of exogenous genes that encode target antigens (J. Natl.
Cancer Inst. 90, 1894-1900, 1998). It is considered that similar
effects can be obtained by the direct injection of untreated DNA
(Annu. Rev. Immunol. 18, 927-974, 2000). In addition, peptide-based
vaccines corresponding to CTL epitopes that directly bind to MHC
molecules, which are safer than DNA-based vaccines, are also under
consideration (Int. J. Cancer 63, 883-885, 1995).
[0010] ATL is a neoplastic disease caused by the infection of
HTLV-I, which shows high carrier rate in Japan, however, it has
been considered to be a malignant tumor with extremely bad
prognosis because of its resistance to chemotherapeutic agents. On
the other hand, various clinical observations indicate the
anti-tumor effect of host cellular immunity, particularly of CTLs.
The object of the present invention is to provide: a screening
method for a CTL recognition antigen or an antigen epitope thereof
which can induce CTLs having an anti-tumor effect against HTLV-I
tumors such as ATL, etc.; a screening method for an adjuvant that
enhances the inducing activity of CTLs having an anti-tumor effect
against HTLV-I tumors by a CTL recognition antigen or an antigen
epitope thereof or an antigen epitope peptide thereof which can
induce CTLs having an anti-tumor effect against HTLV-I tumors; a
immunogenic composition and the like for inducing an immune
response containing said CTL recognition antigen or antigen epitope
thereof, or DNA thereof as an active ingredient.
SUMMARY OF THE INVENTION
[0011] The present inventors made a keen study to attain the object
mentioned above. Splenic T cells derived from syngeneic
immunocompetent rat immunized with nu/+ rat-derived HTLV-I-infected
cell lines, FPM1-V1AX, were repeatedly stimulated every 2 weeks by
using formalin-fixed FPM1-V1AX to establish HTLV-I-specific CTL
cell lines, and the cytotoxic activity of the above-mentioned
HTLV-I-specific CTL cell lines against target cells G14 sensitized
with various synthetic peptides that are considered to include the
dominant recognition epitope of HTLV-I-specific CTLs were studied,
and the present inventors identified a peptide for which target
cells show particularly strong CTL sensitivity, namely the dominant
epitope of a CTL recognition antigen which can induce CTLs having
an anti-tumor effect against HTLV-I tumors. The present inventors
verified that it is possible to induce tumor antigen
epitope-specific CTLs in immunocompetent rats by using synthetic
peptides of said epitope as an immunogen and using an adjuvant, and
that said induced CTLs can strongly suppress proliferation of
HTLV-I-infected tumor cells in vivo. Thus, the present invention
has completed.
[0012] The present invention relates to: a screening method for a
CTL recognition antigen or an antigen epitope thereof, which can
induce cytotoxic T lymphocytes (CTLs) having an anti-tumor effect
against HTLV-I tumors, wherein CTLs induced by a test substance is
administered to a non-human animal model of HTLV-I-associated
disease, and the change of tumors in said non-human animal is
measured and assessed; the screening method for a CTL recognition
antigen or an antigen epitope thereof, which can induce CTLs having
an anti-tumor effect against HTLV-I tumors according to claim 1 or
paragraph 1, wherein the non-human animal model of
HTLV-I-associated disease is a non-human animal model of adult T
cell leukemia; the screening method for a CTL recognition antigen
or an antigen epitope thereof which can induce CTLs having an
anti-tumor effect against HTLV-I tumors according to claim 1 or
paragraph 1, wherein the non-human animal is a rat; a screening
method for a CTL recognition antigen or an antigen epitope thereof
which can induce CTLs having an anti-tumor effect against HTLV-I
tumors, wherein CTLs induced by a test substance is contacted with
HTLV-I-infected tumor cell lines, and the cytotoxic activity of
said CTLs is measured and assessed; a screening method for a CTL
recognition antigen or an antigen epitope thereof which can induce
CTLs having an anti-tumor effect against HTLV-I tumors, wherein
target cells sensitized with a test substance or target cells that
express a test substance are contacted with HTLV-I-specific CTL
cell lines, and the cytotoxic activity of said HTLV-I-specific CTL
cell lines is measured and assessed; the CTL recognition antigen or
the antigen epitope thereof which can induce CTLs having an
anti-tumor effect against HTLV-I tumors, which is obtained by the
screening method of claim 1 or paragraph 1; the antigen epitope
according to claim 4 or paragraph 6, wherein the antigen epitope is
a peptide comprising an amino acid sequence shown by Seq. ID
No.2.
[0013] The present invention further relates to: a screening method
for an adjuvant that enhances an anti-tumor effect against HTLV-I
tumors, wherein CTLs induced by using the CTL recognition antigen
or the antigen epitope thereof according to claim 4 or 5; or
paragraph 6 or 7, and a test adjuvant are administered to a
non-human animal model of HTLV-I-associated disease and the change
of tumors in said non-human animals is measured and assessed; the
screening method for an adjuvant that enhances an anti-tumor effect
against HTLV-I tumors according to claim 6 or paragraph 8, wherein
the non-human animal model of HTLV-I-associated disease is a
non-human animal model of adult T cell leukemia; the screening
method for an adjuvant that enhances an anti-tumor effect against
HTLV-I tumor according to claim 6 or paragraph 8, wherein the
non-human animal is a rat; a screening method for an adjuvant that
enhances an anti-tumor effect against HTLV-I tumors, wherein CTLs
induced by using the CTL recognition antigen or the antigen epitope
thereof according to claims 4 or 5; or paragraph 6 or 7; and a test
adjuvant are contacted with HTLV-I-infected tumor cell lines, and
the cytotoxic activity of said CTLs is measured and assessed.
[0014] The present invention still further relates to: an
immunogenic composition for inducing an immune response containing
a CTL recognition antigen or an antigen epitope thereof which can
induce CTLs having an anti-tumor effect against HTLV-I tumors and
which can be obtained by the screening method according to claim 1
or paragraphs 1-5; an immunogenic composition for inducing an
immune response containing a DNA that encodes a CTL recognition
antigen or an antigen epitope thereof which can induce CTLs having
an anti-tumor effect against HTLV-I tumors obtained by the
screening method according to claim 1 or paragraphs 1-5; the
immunogenic composition for inducing an immune response according
to claim 9 or 10; or paragraph 13 or 14, wherein the CTL
recognition antigen is an HTLV-I Tax protein shown by Seq. ID No.1;
the immunogenic composition for inducing an immune response
according to claim 11 or paragraph 15, wherein the CTL recognition
antigen is a protein comprising an amino acid sequence wherein at
least one amino acid is deleted, substituted, or added in the amino
acid sequence shown by Seq. ID No.1, which can induce CTLs having
an anti-tumor action against HTLV-I tumors; the immunogenic
composition for inducing an immune response according to claim 9 or
10; or paragraph 13 or 14, wherein the CTL recognition antigen
epitope is a peptide comprising the amino acid sequence shown by
Seq. ID No.2; the immunogenic composition for inducing an immune
response according to claim 13 or paragraph 17, wherein the CTL
recognition antigen epitope is a peptide comprising an amino acid
sequence wherein at least one amino acid is deleted, substituted,
or added in the amino acid sequence shown by Seq. ID No.2, which
can induce the CTLs having an anti-tumor action against HTLV-I
tumors; the immunogenic composition for inducing an immune response
according to claims 9 or 10; or paragraph 13 or 14, further
containing an adjuvant that enhances an anti-tumor effect against
HTLV-I tumors; the immunogenic compositon for inducing an immune
response according to claim 15 or paragraph 19, wherein the
adjuvant is obtained by the screening method according to claim 6
or paragraphs 8-12; the immunogenic composition for inducing an
immune response according to claim 15 or paragraph 19, wherein the
adjuvant is ISS-ODN; an HTLV-I recognition CTL induced by the
immunogenic composition for inducing an immune response according
to any of claims 9 to 17 or paragraphs 13 to 21; and a
pharmaceutical composition containing the HTLV-I recognition CTL
according to claim 18 or paragraph 22.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The following Detailed Description, given by way of example,
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the accompanying
drawings, incorporated herein by reference. Various preferred
features and embodiments of the present invention will now be
described by way of non-limiting example and with reference to the
accompanying drawings in which:
[0016] FIG. 1 is a figure showing the results of examining a subset
of immune T cells considered to be directly required for the
regression of tumors in vivo.
[0017] FIG. 2 is a figure showing the results of cytotoxic
activities against target cells of various splenic T cells derived
from rats wherein tumors have been completely regressed.
[0018] FIG. 3 is a figure showing the results of examining viral
antigens recognized by HTLV-I-specific CTLs.
[0019] FIG. 4 is a figure showing the results of inhibition of
cytotoxicity against target cells of HTLV-I-specific CTL cell lines
in the presence of a competitor.
[0020] FIG. 5 is a figure showing the results of cytotoxic
activities of various CTLs induced by HTLV-I-infected tumor cell
line FPM1-V1AX.
[0021] FIG. 6 is a figure showing the results of screening of
HTLV-I-specific CTL cell line recognition peptides.
[0022] FIG. 7 is a figure showing the results of screening of
HTLV-I-specific CTL cell line recognition peptides in the case
where long-tern-cultured CTL cell lines are used.
[0023] FIG. 8 is a figure showing the results of screening 9 amino
acid synthetic peptides recognized by HTLV-I-specific CTL cell
lines.
[0024] FIG. 9 is a figure showing the results of examining the
cytotoxic activity of CTLs against target cells sensitized with
various concentrations of peptides.
[0025] FIG. 10 is a figure showing the specificity of
HTLV-I-specific CTL cell lines against synthetic peptide Tax
180-188.
[0026] FIG. 11 is a figure showing the results of proliferation
suppression effect on HTLV-I tumors in vivo by Tax 180-188
recognition CTL cell lines.
[0027] FIG. 12 is a figure showing the effectiveness of Tax 180-188
and ISS-ODN as immunogenic compositions.
DETAILED DESCRIPTION OF THE INVENTION
[0028] For ease of reference a summary of the accompanying sequence
listings is given below:
[0029] SEQ ID NO:1 shows the amino acid sequence of an HTLV-I Tax
protein.
[0030] SEQ ID NO:2 shows the amino acid sequence of an HTLV-I
anti-tumor CTL recognition epitope.
[0031] SEQ ID NO:3 shows the amino acid sequence of another HTLV-I
anti-tumor CTL recognition epitope.
[0032] SEQ ID NO:4 shows the amino acid sequence of an HTLV-I
anti-tumor CTL recognition epitope.
[0033] SEQ ID NO: 5 shows a nucleic acid sequence of the adjuvant
ISS-ODN.
[0034] SEQ ID NO: 6 shows an amino acid sequence of an influenza A
viral epitope.
[0035] The screening method for a CTL recognition antigen or an
antigen epitope thereof which can induce CTLs having an anti-tumor
effect against HTLV-I tumors of the present invention can be
particularly exemplified by the following: a method wherein CTLs
induced by a test substance are administered to a non-human animal
model of HTLV-I-associated disease such as ATL and the like, and
the change of tumors in said non-human animal is measured and
assessed; a method wherein CTLs induced by a test substance is
contacted with HTLV-I-infected tumor cell lines, and cytotoxic
activity of said CTLs is measured and assessed; a method wherein
target cells sensitized with a test substance or target cells
expressing a test substance is contacted with HTLV-I-specific CTL
cell lines, and the cytotoxic activity of said HTLV-I-specific CTL
cell lines is measured and assessed. As used herein, the CTL
recognition antigen or the antigen epitope thereof, which can
induce CTLs means a CTL recognition antigen or an antigen epitope
thereof which can induce CTLs in vivo and in vitro.
[0036] The above-mentioned non-human animal model of
HTLV-I-associated disease is not particularly restricted as long as
it is a non-human animal which induces HTLV-I-associated diseases
such as ATL, HAM/TSP, HAAP, uveitis, alveobronchiolitis,
sialadenitis akin to Sjogren syndrome, etc., by infection of
HTLV-I. However, the one that can reproducibly proliferate
HTLV-I-infected tumor cells over long-term period is preferable.
Besides, the non-human animal of the present invention can be
particularly exemplified by non-human mammals such as mice, rats,
guinea pigs, monkeys, cats, dogs, horses, cattle, or rabbits, but
they are not limited to these examples. The method of generating a
non-human animal model of ATL will be explained below with an ATL
rat model as an example.
[0037] An ATL rat model can be obtained by, for instance,
administering HTLV-I-infected tumor cell lines to an
immunocompetent rat. However, an ATL rat model which can be
obtained by administering HTLV-I-infected tumor cell lines
subcutaneously, intraperitoneally, intravenously or the like to a
non-human animal which is deficient in T cell function is more
preferable in view of the reproducibility, and the point that
HTLV-I-infected tumor cells can be proliferated and subcultured in
vivo. As for a wild type rat used for evaluation, it is preferable
to use a wild type rat syngeneic to an ATL rat model. The
above-mentioned non-human animal, which is deficient in T cell
function can be particularly exemplified by a nude non-human
animal, including, for example, severe combined immunodeficiency
mice (SCID) and National Institutes of Health nude rat F344/N
Jcl-rnu/rnu, and the like, but they are not limited to these
examples. In addition, there is no limitation to the
above-mentioned HTLV-I-infected tumor cell lines as long as they
are infected with HTLV-I by a known method and of which MHCs
coincide with a wild type non-human animal. For instance, it can be
particularly exemplified by cell lines such as FPM1-V1AX and the
like.
[0038] The test substance used for the screening methods of the
present invention can be particularly exemplified by proteins,
peptides, DNAs, RNAs, antisense DNAs, antisense RNAs, etc. There is
no limitation to the target cells sensitized with the
above-mentioned test substance or the target cells that express a
test substance as long as the MHCs thereof coincide, however, it
can be preferably exemplified by G14 cells, which are CD8.sup.+ T
cell lines (J. Virol. 74, 9610-9616, 2000).
[0039] There is no limitation to the expression system for
preparation of cells that express a test substance as long as the
expression system is capable of expressing the above-mentioned test
substance intracellularly and the examples include, chromosome-,
episome-, and virus-derived expression systems, for instance,
vectors derived from bacterial plasmid, vectors derived from yeast
plasmid, vectors derived from papovavirus such as SV40, vaccinia
virus, adenovirus, fowlpox virus, pseudorabies virus, retrovirus,
and vectors derived from bacteriophage or transposon and vectors
derived from the combination of these two, e.g. vectors derived
from genetic factors of plasmid and bacteriophage, such as cosmid
and phagemid. This expression system may contain a regulatory
sequence that not only induces expression but also regulate the
expression. In addition, an expression vector series, which is
capable of translating by changing the reading frame can also be
used advantageously.
[0040] The expression system wherein said test substance is
incorporated can be introduced into a host cell by the methods
described in many standard laboratory manuals such as manuals of
Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and of
Maniatis et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989), and can be conducted by the examples including calcium
phosphate transfection, DEAE-dextran-mediated transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape loading, ballistic
introduction, infection, etc.
[0041] There is no particular limitation to the HTLV-I-specific CTL
cell lines of the present invention as long as they are the cell
lines that specifically recognize HTLV-I, however, the cell lines
restrained by MHC class I are more preferable. Said HTLV-I-specific
CTL cell lines restrained by MHC class I can be obtained by a known
method, for instance, a method wherein non-human animal-derived T
cells that express MHC class I molecules are infected with HTLV-I,
and proliferated in the body of a non-human animal which is
deficient in T cell function (nude non-human animal), to establish
HTLV-I-infected tumor cell lines, then the splenic T cells of the
immunocompetent non-human animal which is immunized with such
established cell lines are repeatedly stimulated with
formalin-fixed HTLV-I-infected tumor cell lines.
[0042] The CTL recognition antigen which can be obtained by the
above-mentioned screening method and which is capable of inducing
CTLs having an anti-tumor effect against HTLV-I tumors can be
exemplified by an HTLV-I Tax protein shown by Seq. ID No.1, a
protein comprising an amino acid sequence wherein at least one
amino acid is deleted, substituted, or added in the amino acid
sequence shown by Seq. ID No.1 and which is capable of inducing
CTLs having an anti-tumor action against HTLV-I tumors or the like.
The antigen epitope which can be obtained by the above-mentioned
screening method can be particularly exemplified by a peptide
comprising an amino acid sequence shown by Seq. ID Nos.2, 3, or 4,
or particularly preferably, a peptide comprising an amino acid
sequence shown by Seq. ID No.2, a peptide comprising an amino acid
sequence wherein at least one amino acid is deleted, substituted,
or added in these amino acid sequences and which is capable of
inducing CTLs having an anti-tumor action against HTLV-I tumors or
the like, however, such antigen epitopes are not limited to these
examples.
[0043] Therefore, the invention further comprehends polynucleotides
that express such homologous polypeptides; and the corresponding
degrees of homology or identity of those polynucleotides to
polynucleotides encoding polypeptides to which homologous
polypeptides have homology or identity of at least 50% to 100%.
Homologous polypeptides advantageously contain one or more epitopes
of the polypeptide to which there is identity or homology, such
that homologous polypeptides exhibit immunological similarity or
identity to the polypeptide to which there is identity or homology,
e.g., the homologous polypeptide elicits similar or better immune
response (to the skilled immunologist) than polypeptide to which
there is identity or homology and/or the homologous polypeptide
binds to antibodies elicited by and/or to which the polypeptide to
which there is identity or homology binds, advantageously and not
to other antibodies.
[0044] Accordingly, fragments of homologous polypeptides and of
polypeptides to which there is identity or homology, advantageously
those fragments which exhibit immunological similarity or identity
to homologous polypeptides or polypeptides to which there is
identity or homology, are envisoned as being expressed, and
therefore, polynucleotides therefor which may represent fragments
of polynucleotides of homologous polypeptides and of polypeptides
to which there is identity or homology, are also envisioned by and
useful in the instant invention.
[0045] The screening method for an adjuvant that enhances an
anti-tumor effect against HTLV-I tumors of the present invention,
namely, an adjuvant which is capable of inducing CTLs specific to a
CTL recognition antigen or an antigen epitope thereof having a more
efficient anti-tumor effect against HTLV-I tumors is not
particularly restricted as long as such method includes: a method
wherein CTLs induced by using the above-mentioned CTL recognition
antigen or an antigen epitope thereof, or preferably, an antigen
epitope peptide comprising the amino acid sequence shown by Seq. ID
No.2 and a test adjuvant are administered to a non-human animal
model of HTLV-I-associated disease such as adult T cell leukemia
non-human animal model and the like, and the change of tumors in
said non-human animal is measured and assessed; a method wherein
CTLs induced by using the above-mentioned CTL recognition antigen
or the antigen epitope thereof, or preferably, CTLs induced by
using an antigen epitope peptide comprising the amino acid sequence
shown by Seq. ID No.2 and a test adjuvant is contacted with
HTLV-I-infected tumor cell lines, and cytotoxic activity of said
CTLs is measured and assessed. The adjuvant which can be obtained
by such screening method can be particularly exemplified by the
following examples: ISS-ODN that includes CpG motif which is
capable of efficiently inducing peptide-specific CTLs
(Immunostimulatory DNA sequences-oligodeoxynucleotide; Nat. Med. 3,
849-854, 1997), QS21 that stimulates cytotoxic T cells (Quillaia
saponaria; which can be commercially obtained from Cambridge
Biotech, Worcester, Mass.), aluminum hydroxide, aluminum phosphate,
aluminum oxide, oil-based emulsions, complete and incomplete
Freund's adjuvant, saponin, vitamin E lysate, RIBI, ISCOM, among
others.
[0046] The CTL recognition antigen or the antigen epitope thereof,
or DNA encoding them which can be obtained by the aforementioned
screening method and which can induce CTLs having an anti-tumor
effect against HTLV-I tumors can be used as a immunogenic
composition for inducing an immune response such as cellular
immunity, humoral immunity and the like. The immunogenic
composition for inducing an immune response of the present
invention can be exemplified by a CTL recognition antigen which can
induce CTLs having an anti-tumor effect against HTLV-I tumors, of
which examples include: an HTLV-I Tax protein shown by Seq. ID
No.1; a protein comprising an amino acid sequence wherein one or a
few amino acids are deleted, substituted, or added in the amino
acid sequence shown by Seq. ID No.1 and which can induce CTLs
having an anti-tumor action against HTLV-I tumors, or a CTL
recognition antigen epitope, of which examples include: an
immunogenic composition for inducing an immune response containing
a peptide comprising the amino acid sequence shown by Seq. ID No.2,
or a peptide comprising an amino acid sequence wherein one or a few
amino acids are deleted, substituted, or added in the amino acid
sequence shown by Seq. ID No. 2, and which can induce CTLs having
an anti-tumor action against HTLV-I tumors as an active ingredient,
or an immunogenic composition for inducing an immune response
containing DNA that encodes the above-mentioned CTL recognition
antigen and the antigen epitope thereof which can induce CTLs
having an anti-tumor action against HTLV-I tumors as an active
ingredient. As for the immunogenic composition for inducing an
immune response of the present invention, the one containing
various adjuvants that further enhances cellular or local immunity
is preferable. Said adjuvant can be exemplified by the one can be
obtained by a screening method for an adjuvant that enhances an
anti-tumor effect against the aforementioned HTLV-I tumors such as
ISS-ODN and the like. When an adjuvant is used, it can also be used
as a recombinant fusion protein or a recombinant fusion peptide
generated from DNA which continuously encodes various fungus
components, toxins and the like to be used as an adjuvant together
with the above-mentioned CTL recognition antigen or an antigen
epitope thereof, preferably an antigen epitope peptide comprising
the amino acid sequence shown by Seq. ID No.2.
[0047] The immunogenic composition for inducing an immune response
of the present invention may contain a pharmaceutically acceptable
carrier or a diluent, an immunostimulator, an additive agent, etc.
The carrier or diluent can be particularly exemplified by the
following: a stabilizing agent such as SPGA; carbohydrates such as
sorbitol, mannitol, starch, sucrose, glucose, dextran and the like;
proteins such as albumin, casein and the like; protein-containing
substances such as bovine sera, skim milk and the like; buffer
solutions such as phosphate buffer solution, physiological saline,
water and the like. The immunostimulator can be particularly
exemplified by cytokines such as interleukin-2 (IL-2),
interleukin-12 (IL-12), tumor necrosis factor .alpha. (TNF-.alpha.)
and the like. The additive agent can be exemplified by polypeptides
of low molecular weight (less than approximately 10 residues),
proteins, amino acids, carbohydrate containing glucose or dextran,
chelating agents such as EDTA and the like, protein stabilizing
agents, inhibitors or suppressors of microorganism proliferation
and the like, but they are not limited to these examples.
[0048] The pharmaceutical agents or pharmaceutical compositions
provided by the present invention can be exemplified by the one
containing HTLV-I recognition CTLs induced by administering a
immunogenic composition for inducing an immune response to
non-human animals and the like as an active ingredient, as well as
the above-mentioned immunogenic composition for inducing an immune
response. Preferable forms for these pharmaceutical agents or
pharmaceutical composition are the forms which can be administered
orally, intravenously, intraperitoneally, intranasally,
intracutaneously, subcutaneously, intramascularly and the like.
Effective doses to be administered can be determined accordingly by
considering the types and compositions of such pharmaceutical
agents or pharmaceutical compositions, the administration methods,
the age and body weight of a patient, etc. It is preferable to
administer them one or a few times a day. When administered orally,
it is ordinarily administered through the formulations prepared by
mixing with a carrier for formulation. Here, a substance which is
usually used in the formulation field, and which does not react
with the peptide of the present invention is used as a carrier for
formulation.
[0049] Further, dosage forms can be particularly exemplified by
tablets, capsules, granules, powder, syrup, suspension agent,
suppository, ointment, cream, gel, patch, inhalant, injectable
solution, etc. These formulations are prepared by ordinary
protocols, and particularly, liquid formulation can be dissolved or
suspended into water or other appropriate media when used. Tablets
and granules may be coated by a known method. Injectable solution
is prepared by dissolving the peptide of the present invention into
water, however, it may also be dissolved into physiological saline
or glucose solution according to need, and buffer agent or
preservation agent may be further added. These formulations may
also contain other ingredients of therapeutic value.
[0050] The composition may optionally comprise a pharmaceutically
acceptable carrier, diluent, excipient or adjuvant. The choice of
pharmaceutical carrier, excipient or diluent can be selected with
regard to the intended route of administration and standard
pharmaceutical practice. The pharmaceutical compositions may
comprise as (or in addition to) the carrier, excipient or diluent,
any suitable binder(s), lubricant(s), suspending agent(s), coating
agent(s), solubilising agent(s), and other carrier agents that may
aid or increase entry of the pharmaceutical composition or agent
into a preferred tissue site (such as for example a lipid delivery
system).
[0051] The CTL recognition antigen or the antigen epitope thereof
which can induce CTLs having an anti-tumor effect against HTLV-I
tumors of the present invention can also be ingested as a
functional food by compounding them into the following as food
materials for infection control of HTLV-I and/or symptom
improvement of HTLV-I-associated disease: baked goods either frozen
or fresh such as puddings, chocolates, cookies, breads, cakes,
jellies, crackers, and including pizza, biscuits, pies etc.;
confectioneries such as sweet jellied azuki-bean pastes, etc.;
breads and confectioneries such as frozen desserts, chewing gums,
etc; noodles such as wheat noodles, buckwheat noodles, etc.; fish
paste products such as steamed fish pastes, hams, fish meat
sausages; various beverages such as yogurts, yogurt drinks, juice,
cow milk, soy milk, alcoholic beverages, coffee, tea, natural leaf
tea, oolong tea, isotonic drinks, etc.; condiments such as soybean
pastes, soy sauce, dressing, mayonnaise, edulcorant, etc.;
bean-curds; devil's tongue; various prepared food such as fish
boiled in soy sauce, dumplings, croquettes, salads, nuts; dairy
products including cheese, whipped desserts, and ice cream; meat
products including sausages, fish, ham, pork and beef, such as
joints of pork or beef; fresh and dried fruit; and snacks.
[0052] Similarly, the antigen or antigen epitopes can be added to
compositions that can be combined with animal feed stock and/or
water provisions, dog food, cat food, bird food, or rodent food.
One of skill in the art will understand this method of
administration is sometimes referred to as "bait dropping," in
which the pharmaceutical composition is included within the food
and/or water of the organism to be vaccinated.
INDUSTRIAL APPLICABILITY
[0053] CTLs induced by using protein, peptide, nucleic acid and the
like of a CTL recognition antigen or an antigen epitope thereof
which can induce CTLs having an anti-tumor effect against HTLV-I
tumors as an immunogen, and by using an adjuvant obtained from the
screening method of the present invention can strongly suppress the
proliferation of HTLV-I-infected tumor cells in vivo. Therefore,
protein, peptide, nucleic acid and the like of such CTL recognition
antigen or such antigen epitope thereof which can induce CTLs
having an anti-tumor effect against HTLV-I tumors are useful as a
immunogenic composition for HTLV-I-associated diseases such as an
adult T cell leukemia (ATL), etc., and CTLs induced by said
protein, peptide, nucleic acid and the like are useful as a
preventive and therapeutic agent for the above-mentioned
HTLV-I-associated diseases.
[0054] The present invention will be further particularly explained
hereinafter with reference to the examples, but the scope of the
invention will not be limited to these examples.
EXAMPLES
Example 1
Cell Lines
[0055] FPM1-V1AX (J. Virol. 73, 6031-6040, 1999) was established by
proliferating immortalized rat T cell lines FPM1 established
through the infection of HTLV-I with T cells of immunocompetent
rats F344/N Jcl-rnu/+ (nu/+) (4-week-old female rats; Clea Japan,
Inc.), in the body of a nude rat F344/N Jcl-rnu/rnu (nu/nu) (J.
Virol. 73, 6436-6443, 1999). FPM-SV was established by transforming
HTLV-I negative SV40 to nu/+rat-derived renal cell lines (J. Virol.
73, 6031-6040, 1999). An HTLV-I-infected T cell line established
from splenocytes of WKAH rats (TARS-1; J. Exp. Med. 159, 1105-1116,
1984) was kindly provided by Dr. T. Yoshiki of Hokkaido University
and used in the experiment. RT1.A.sup.1/TARS-1 was established by
transfecting RT1.A.sup.1-expressing plasmid pRep10 into TARS-1
cells followed by in vitro selection with 400 .mu.g/ml of
hygromycin. Besides, pRep10 (Immunogenetics 39, 447, 1994) was
kindly provided by Dr. S. Salgar (Miami Univ., FL) and used herein.
The expression of RT1.A.sup.1 in the above-mentioned
RT1.A.sup.1/TARS-1 cells was confirmed by immunofluorescence
analysis. G14-Tax cell lines which are the cells that stably
express G14 and Tax genes, i.e. the IL-2-dependent HTLV-I negative
CD8.sup.+ T cell lines established from nu/+rats were prepared by
the method as described previously (J. Virol. 74, 9610-9616, 2000).
All the cell lines used herein were maintained in RPMI1640
containing 10% heat-inactivated FCS (Whittaker, Walkersville, Md.),
penicillin, and streptomycin. 10 U/ml of recombinant human IL-2
(rhIL-2) (Shionogi Pharmaceutical & Co., Ltd.) was added to the
culture medium RPMI1640 for maintaining G14 and G14-Tax.
Example 2
Preparation of Immune T cell Subset
[0056] 4-week-old nu/+ rats were immunized with intraperitoneal
administration of 2.times.10.sup.7 of FPM1-V1AX cells two times
with a two-week interval. One week after the last immunization,
splenic T cells were isolated and purified through a nylon wool
column, then lymphocytes subsets were further purified using
complement lysis method. To explain briefly, supernatants of
splenic T cells were mixed with anti-rat CD8 monoclonal antibodies
(R1-10B5) or anti-rat CD4 monoclonal antibodies (RTH-7), and
incubated on ice for 30 minutes. After washed with 1% FCS-PBS, the
above-mentioned cells were incubated in the culture medium RPMI1640
containing 2% rabbit serum (Cedarlane Laboratories Limited,
Ontario, Canada) at 37.degree. C. for 45 minutes, then washed two
times with 10% FCS-RPMI 1640. Subsequently, it was confirmed by a
flow cytometry analysis whether CD8.sup.+ or CD4.sup.+ T cells were
condensed to on and above 98%.
Example 3
Anti-Tumor Effect of CD4+ or CD8.sup.+ T Cells
[0057] The present inventors previously reported that adoptive
transfer of splenic T cells from rats immunized with
HTLV-I-infected cells effectively inhibited the proliferation of
malignant lymphomas in F344/N Jcl-rnu/rnu (nu/nu) rats wherein an
HTLV-I-infected tumor cell line FPM1-V1AX was administered (J.
Virol. 73, 6031-6040, 1999). Therefore, in order to clarify which
subsets of immune T cells are directly required for tumor
regression in vivo, anti-tumor effect against each subset of F344/N
Jcl-rnu/rnu (nu/nu) rats was examined by using CD4+ or CD8.sup.+
cell groups isolated by the method described in Example 2.
2.times.10.sup.7 of FPM1-V1AX cell lines were subcutaneously
inoculated into 4-week-old nu/nu female rats (Clea Japan, Inc.)
that were simultaneously administered intraperitoneally with 107 of
CD4.sup.+ T cells (.tangle-solidup.), CD8.sup.+ T cells
(.box-solid.), or total T cells (.circle-solid.) isolated in
Example 2. Then suppressing effect on tumor proliferation was
examined by measuring the size of each subcutaneous tumor with a
caliper every other day. In these measurements, the longest surface
length (mm; a) and width (mm; b) were determined by the
above-mentioned measurement, and the tumor volume (V; mm.sup.3) was
calculated using the formula (V=ab.sup.2/2) as described previously
(J. Virol. 73, 6031-6040, 1999). Besides, 4-week-old nu/nu rats
subcutaneously inoculated with FPM1-V1AX cell lines alone served as
controls (.largecircle.).
[0058] The results of the above are shown in FIG. 1. It was
confirmed that both CD4+ and CD8.sup.+ T cells exhibited equivalent
inhibitory effect on the growth of HTLV-I-infected tumor cell lines
as well as total T cells. No metastatic tubercles were observed in
the rats which underwent transfer of any subsets of the immune T
cells, in contrast to the visible tubercles in the lungs and
mediastinal lymph nodes of tumor-bearing rats.
[0059] In order to conduct further analysis, splenic T cells
(CD4.sup.+ T cells, CD8.sup.+ T cells, or total T cells) were
isolated respectively from each nu/nu rat on day 28 when complete
tumor regression occurred by transferring immune T cells (CD4.sup.+
T cells, CD8.sup.+ T cells, or total T cells), then their cytotoxic
activities against the respective target cells were examined. The
above-mentioned various splenic T cells (5.times.10.sup.6
cells/well) were cocultured with formalin-fixed FPM1-V1AX cells
(2.times.10.sup.6 cells/well) in 2 ml of 10% FCS-RPMI 1640 per well
of a 24-well plate for 6 days. Cytotoxic activities against target
cells were measured by .sup.51Cr release assay for 6 hours as
described previously (Immunology 14, 181-196, 1968) at the ratio of
said cells (E; effector cells) and target cells (T; FPM1-V1AX,
syngeneic T cell lines G14, or G14-Tax) (E/T) as 10, and calculated
by the formula as described below. In addition, values (mean.+-.SD)
were evaluated from three independent experiments. 1 [ experimental
51 Cr release - spontaneous 51 Cr release ] [ maximum 51 Cr release
- spontaneous 51 Cr release ] .times. 100 % Formula 1
[0060] The results of the above are shown in FIG. 2. From these
results, various T cells showed high cytotoxic activities against
FPM1-V1AX and G14-Tax, however, they did not show a cytotoxic
activity against HTLV-I negative G14 cells. In addition, it was
notably recognized by flow cytometry that the splenic T cells
isolated from nu/nu rats wherein the above-mentioned various immune
T cells (CD4+ or CD8.sup.+ immune T cells) were transferred, were
positive for CD4 or CD8 respectively. These results indicate that
an HTLV-I-specific cytotoxic activity of CD4+ or CD8.sup.+ T cells
is strongly involved in direct elimination of HTLV-I-infected tumor
cells in vivo.
Example 4
Recombinant Vaccinia Virus
[0061] Subsequently, in order to examine viral antigens recognized
by HTLV-I-specific CTLs, the following recombinant vaccinia viruses
containing 4 types of HTLV-I genes provided by Dr. H. Shida,
Hokkaido University (rvv; Embo J. 6, 3379-3384, 1987; J. Virol. 62,
3718-3728, 1988; Cell 55, 197-209, 1988) were used herein: rvv
containing HTLV-I env gene (WR-env); rvv containing HTLV-I gag gene
(WR-gag); rvv containing HTLV-I pX gene (WR-40X and WR-27X). WR-27X
is a recombinant vaccinia virus that expresses p21X, p27rex, and
p40tax, whereas WR-40X is the one that expresses p21X and p40tax.
Each of the above-mentioned HTLV-I-rvv (WR-40X, WR-27X, WR-env,
WR-gag) was infected with FPM-SV cells at an m.o.i. (multiplicity
of infection) of 10 at 37.degree. C. for 1 hour. After incubation,
the cells were washed once with 10% FCS-RPMI 1640, then cultured in
10% FCS-RPMI 1640 at 37.degree. C. for 12 hours. Said infected
cells were labeled with a radioactive isotope in order to be used
for the following example.
Example 5
HTLV-I Antigen Recognized by Rat CTLs
[0062] Splenic T cells isolated from nu/+ rats that had been
intraperitoneally administered with FPM1-V1AX were stimulated with
formalin-fixed FPM1-V1AX for 6 days, and then used as effector
cells in the following example. As the target cells, FRM1-V1AX,
FPM-SV, and various HTLV-I-rvv-infected FPM-SV cells (WR-40X,
WR-27, WR-env, WR-gag) prepared in Example 4 were used. In
addition, the cells wherein rvv which does not contain an HTLV-I
gene are infected with FPM-SV cells (WR-HA) were used as controls.
As a result of immunofluorescence analysis conducted for the
above-mentioned HTLV-I-rvv-infected FRM-SV cells, these cells
showed positive for MHC class I, but negative for MHC class II
antigen. Therefore, these cells are considered to selectively
express an antigen confined to MHC class I.
[0063] In the same way as described in Example 3, .sup.51Cr release
assay was conducted using the above-mentioned effector cells (E)
and target cells (T) at each E/T ratio as shown in FIG. 3.
Measurements (mean.+-.SD) were evaluated from three independent
experiments. The results are shown in FIG. 3. These results show
that effector cells have high cytotoxicity against FPM1-V1AX, but
not FPM-SV cells. Among HTLV-I-rvv-infected FPM-SV cells, WR40X-
and WR-27X-infected cells, which commonly express HTLV-I tax,
showed the highest sensitivity for effector cells. The target cells
that express HTLV-I env (envelope) also showed sensitivity, but to
a lesser degree. However, cytotoxic activities against FPM-SV cells
that express HTLV-I gag were as low as the cytotoxic activities
against target cells (WR-HA) as controls.
[0064] Subsequently, the inhibition of cytotoxicity of
HTLV-I-specific CTL cell lines against target cells by unlabeled
cells was examined. The CD8.sup.+ CTL cell lines which are more
specific to HTLV-I Tax obtained by the long-term culture and the
target cells ([.sup.3H]-TdR-labeled FPM1-V1AX:5.times.10.sup.3
cells/well) were plated on 96-well U-bottom plates at an E/T ratio
of 10, then a competitor [unlabeled FPM1-V1AX (.circle-solid.),
G14-Tax (.box-solid.), or G14 (.tangle-solidup.)] was added at a
competitor-to-target ratio as described in FIG. 4, and incubated at
37.degree. C. for 6 hours. Thereafter, cells were harvested with a
Micro 96 Harvester (Skatron), then the amount of remaining target
cells was measured by a microplate .beta.-counter (Micro Beta
Plus), and the specific cytotoxicity (%) was calculated by the
following formula. Values (mean.+-.SD) were evaluated from three
independent experiments. The cells were incubated with 3.7 MBq of
[.sup.3H]-TdR per 10.sup.6 cells at 37.degree. C. for 12 hours,
then washed three times in advance were used as the above-mentioned
target cells. 2 [ cpm without the presence of an effector - cpm in
the presence of an effector ] [ cpm without the presence of an
effector ] .times. 100 % Formula 2
[0065] The results of the above are shown in FIG. 4. These results
show that cytotoxic activities of CTL cell lines against FPM1-V1AX
are notably inhibited by increasing unlabeled G14-Tax but not G14
cells. These results indicate that HTLV-I Tax is one of the major
antigen specifically recognized by CTLs derived from nu/+ rats
administered with syngeneic HTLV-I-infected cells in vivo.
Example 6
MHC Class I Restrain of HTLV-I-Specific Cytotoxic Activity in nu/+
Rats
[0066] For induction of HTLV-I-specific CTL cell lines (two types
of CD8.sup.+ CTL cell lines and CD4.sup.+ CTL cell lines) in
long-term culture, 2.5.times.10.sup.6 cells/well of splenic T cells
were cocultured with the same number of formalin-fixed FPM1-V1AX
cells in 10% FCS-RPMI 1640 wherein 20 U/ml of rhIL-2 was added,
with periodical stimulation using formalin-fixed FPM1 cells every
two weeks, and two types of CD8+CTL cell lines (CD8.sup.+ CTL-1 and
CD8.sup.+ CTL-2) and CD4.sup.+ CTL cell lines were established.
Then, the MHC class I restrain against cytotoxic activities was
examined in the same way as Example 5 using such induced
HTLV-I-specific CTL cell lines. As target cells, 4 cell lines
labeled with [.sup.3H]-TdR in the same way as in Example 5 were
used: nu/+rat-derived FPM1-V1AX that expresses a rat MHC class I
molecule, RT1.A.sup.1 X; WKAH rat-derived W7KSV that does not
express RT1.A.sup.1; WKAH rat-derived TARS-1 that does not express
RT1.A.sup.1; a cell line RT1.A.sup.1/TARS-1 established by stably
transfecting RT1.A.sup.1 cDNA to TARS-1 cells.
[0067] The results of the above are shown in FIG. 5. Two types of
CD8.sup.+ CTL cell lines (CD8.sup.+ CTL-1 and CD8.sup.+ CTL-2)
notably dissolved RT1.A.sup.1/TARS-1 cell but not TARS-1 cells,
however, dissolution was not recognized in CD4.sup.+ CTL cell lines
derived from nu/+ rats immunized with FPM1-V1AX cells. These
findings show that the cytotoxic activity of CD8.sup.+ CTL cell
lines specific to nu/+rat-derived HTLV-I Tax is restrained by
RT1.A.sup.1 of rat MHC class I.
Example 7
Identification of Recognition Epitope of HTLV-I-Specific CTLs
[0068] Peptide mapping analysis was conducted to identify target
epitopes recognized by HTLV-I Tax-specific CD8.sup.+ CTLs obtained
from nu/+ rats immunized with FPM1-V1AX. HTLV-I-specific CTL cell
lines restrained by rat MHC class I (RT1.A.sup.1) were established
by repeating stimulation with formalin-fixed FPM1-V1AX every two
weeks to splenic T cells derived from syngeneic immunocompetent
rats immunized with nu/+ rat-derived HTLV-I-infected cell lines
FPM1-V1AX. Some of a series of synthetic peptides corresponding to
the amino acid sequences of HTLV-I Tax shown in FIGS. 6, 7 and 8
were synthesized using a solid phase peptide synthesis method on an
automated peptide synthesizer (model PSSM-8; Shimazu Corporation,
Kyoto, Japan) with chemicals and program cycles provided by the
manufacturer, then separated from plastic using hydrogen fluoride,
and purified to on and above 95% purity by a reverse-phase
chromatography on an HPLC system (Model SPRINT; PE Biosystems
Japan, Tokyo, Japan). In addition, 9 mer oligopeptides (Tax
179-187, 180-188, 181-189, 182-190, 183-191, 184-192, 185-193,
186-194, 187-195, and influenza Matrix 58-66) were synthesized by
Hokudo Co. (Hokkaido, Japan) on commission and used herein.
[0069] For sensitization of the target cells to be used for the
cytotoxic activity analysis, 29 partially overlapping synthetic
peptides (15-24 mers; partial peptide in the Tax proteins shown in
FIGS. 6 and 7) were added to target cells derived from syngeneic
rats labeled with a radioactive isotope [.sup.3H]-TdR (G14 cells;
5.times.10.sup.3 cells/well) to a concentration of 10 .mu.M each,
and cultured at 37.degree. C. for 1 hour, then the sensitivity for
HTLV-I-specific CTL cell lines was measured by [.sup.3H]-TdR
release assay for 6 hours. Values (mean.+-.SD) were evaluated from
three independent experiments. In addition, E/T was set at 10. The
results are shown in FIGS. 6 and 7. These results show that the
HTLV-I-specific CTL cell lines effectively dissolve target cells
sensitized with peptide Tax 177-200 or Tax 181-195, and show low
cytotoxic activity against target cells sensitized with peptide Tax
17-40, Tax 33-56, Tax 81-104, or Tax 97-120 (FIG. 6). It was
confirmed that long-term-cultured CTL cell lines also notably
dissolved target cells sensitized with Tax 177-200 or Tax 181-195
(FIG. 7). Significant cytotoxicity was not recognized in target
cells sensitized with synthetic peptides other than Tax 177-200 or
Tax 181-195.
[0070] Based on the above-mentioned results, 10 different types of
9 amino acid synthetic peptides (9m peptide) included in the region
of Tax 177-200 were synthesized, and detailed mapping of HTLV-I
Tax-specific CTL epitope was conducted in the same method as
mentioned above. The amino acid sequences of synthetic peptides
used herein are shown in Table 1, and the results of cytotoxic
activity analysis using these 9 amino acid synthetic peptides are
shown in FIG. 8. These results show that the target cells
sensitized with peptide Tax 180-188, Tax 181-189 show particularly
strong CTLs sensitivity, then the target cells sensitized with Tax
179-187, Tax 182-190 exhibited CTL sensitivity for CTLs, however,
the effect of Tax 179-187 varies among multiple experiments. The
above-mentioned results indicated the possibility of the presence
of the dominant epitope recognized by HTLV-I-specific CTLs in Tax
180-188 to Tax 182-190.
1 TABLE 1 Peptide Amino acid sequence Tax 177-200
GQLGAFLTNVPYKRIEELLYKISL Tax 181-195 AFLTNVPYKRIEELL Tax 179-187
LGAFLTNVP Tax 180-188 GAFLTNVPY Tax 181-189 AFLTNVPYK Tax 182-190
FLTNVPYKR Tax 183-191 LTNVPYKRI Tax 184-192 TNVPYKRIE Tax 185-193
NVPYKRIEE Tax 186-194 VPYKRIEEL Tax 187-195 PYKRIEELL Tax 190-198
RIEELLYKI Tax 11-19 LLFGYPVYV (control peptide)
[0071] Subsequently, the peptide concentrations of Tax 180-188, Tax
181-189, Tax 182-190 necessary for inducing cytotoxic activity of
CTLs were examined in order to clarify the dominant recognition
epitope. Target cells G14 treated respectively with serially
diluted peptides for 1 hour in advance, were cocultured with
HTLV-I-specific CTL cell lines, and the CTL sensitivities of target
cells were examined. The E/T ratio was at 10, and values
(mean.+-.SD) were evaluated from three independent experiments. The
results are shown in FIG. 9. These results show that strong CTL
sensitivity was confirmed in target cells sensitized with Tax
180-188 (.circle-solid.), and they show sensitivity to CTL even in
extremely low peptide concentration, i.e. 10-.sup.3 pM. On the
other hand, it was revealed that the concentration of 1 .mu.M for
Tax 181-189 (.box-solid.), and 10 .mu.M for Tax 182-190
(.tangle-solidup.) were necessary to induce cytotoxic activity of
the same extent as Tax 180-188. No CTL sensitivity was recognized
at any concentration in Tax 11-19 (.largecircle.), which was used
as a control therein. The above-mentioned results revealed that the
dominant recognition epitope of HTLV-I-specific CTLs restrained by
RT1.A.sup.1 was a peptide comprising 9 amino acids, i.e. Tax
180-188 (GAFLTNVPY: Seq. ID No.2).
Example 8
Recognition of Tax 180-188 by Tax-Specific CTLs of HTLV-I
[0072] In order to examine whether the peptide Tax 180-188 is
dominant among the target epitopes in HTLV-I Tax recognized by
HTLV-I-specific CTL cell lines, the cytotoxic activities of HTLV-I
Tax-specific CTL cell lines against [.sup.3H]-TdR-labeled G14-Tax
cell were measured in the presence of G14 cells and unlabeled
G14-Tax cells (.circle-solid.) which had been sensitized in advance
with unlabeled competitors such as, untreated G14 cells
(.largecircle.), 10 .mu.M of Tax 180-188 (.box-solid.), or Tax
11-19 (.tangle-solidup.). The E/T ratio was at 10, and values
(mean.+-.SD) were evaluated from three independent experiments. The
results are shown in FIG. 10. As a result, it was revealed that
when G14 cells treated with 10 .mu.M of Tax 180-188 (.box-solid.)
were used, the cytotoxic activities against radiolabeled G14-Tax
were completely inhibited when the competitor/Target cells ratio
was at 40. The competitive effect of G14 cells treated with Tax
180-188 was at the same extent as the competitive effect of
unlabeled G14-Tax (.circle-solid.). Untreated G14 cells
(.largecircle.) or G14 cells treated with 10 .mu.M of various
peptides [Tax 181-189, Tax 182-190, and Tax 11-19
(.tangle-solidup.)], scarcely affected the cytotoxic activities of
HTLV-I Tax-specific CTL cell lines. These results indicate that the
epitope Tax 180-188 is the dominant epitope recognized by HTLV-I
Tax-specific CTL cell lines.
Example 9
Suppression Effect on Proliferation of HTLV-I-infected Tumor Cells
of Tax 180-188 Recognition CTL Cell Lines In Vivo
[0073] It was examined whether Tax 180-188 recognition CTL cell
lines can possibly suppress the proliferation of HTLV-I-infected
tumor cell FPM1-V1AX in rats in vivo. Suppression effect on tumor
proliferation in rats wherein FPM1-V1AX was inoculated
subcutaneously and CTL cell lines (107) that recognize Tax 180-188
were simultaneously administered intraperitoneally
(.tangle-solidup.) was observed by using nu/nu female rats
subcutaneously inoculated with HTLV-I-infected tumor cell FPM1-V1AX
(2.times.10.sup.7) alone as positive controls. Besides, the change
of tumor proliferation was measured by measuring volume of each
subcutaneous tumor by a caliper every other day as in Example 3,
and the tumor volumes (V; mm.sup.3) were thus measured. Time
courses of these subcutaneous tumor proliferations are shown in
FIG. 11. The results show that subcutaneous tumors continued to
proliferate in rats inoculated with FPM1-V1AX alone
(.circle-solid.), whereas such proliferation was promptly rejected
in rats simultaneously transfected with CTLS (.tangle-solidup.). As
a result of the above-mentioned, it was indicated that Tax 180-188
recognition CTL cell lines can possibly suppress in vivo
proliferation of HTLV-I-infected tumor cells. In other words, there
is a possibility that Tax 180-188 peptide is an important epitope
as a tumor rejection antigen.
Example 10
Vaccine Effect of Tax 180-188 and ISS-ODN
[0074] Whether the antigen peptide determined as above-mentioned
has actually become a tumor rejection antigen alone in vivo is
extremely important in considering to what extent this antigen
peptide can be applied as a tumor vaccine model. Subsequently,
therefore, it was considered whether a peptide vaccine can induce
Tax 180-188-specific CTLs in vivo, and whether such induced CTLs
can show an anti-tumor effect in vivo. ISS-ODN (Immunostimulatory
DNA sequences-oligodeoxynucleotide) containing CpG motif was used
as an adjuvant, in order to induce Tax 180-188-specific CTLs more
efficiently (Nat. Med. 3, 849-854, 1997). In addition, as an
ISS-ODN [ESPEC OLIGO SERVICE; 5'-TGACTGTGAACGTTCGAGATGA-3- ' (Seq.
ID No.5)], the one synthesized as phosphorothioate single-stranded
oligonucleotides were used. 100 .mu.g of Tax 180-188 synthetic
peptide (.box-solid.), 10 mmol of ISS-ODN (.tangle-solidup.), 100
.mu.g of Tax 180-188 mixed with 10 nmol of ISS-ODN (*), or 100
.mu.g of Influenza A matrix (GILGFVFTL; Seq. ID No.6) peptide
comprising the 58-66.sup.th amino acid sequences mixed with 10 nmol
of ISS-ODN (.diamond-solid.) were respectively mixed in 200 .mu.l
of physiological saline and administered subcutaneously into
4-week-old nu/+ female rats, and they were immunized again after
two weeks on the same condition. Besides, rats subcutaneously
administered with physiological saline alone were used as controls
(.circle-solid.). Two weeks after the last immunization, splenic T
cells (107) were separated from each rat, then administered
intraperitoneally to 4-week-old nu/nu female rats, that were
simultaneously administered intraperitoneally with HTLV-I-infected
tumor cells, FPM1-V1AX (2.times.10.sup.7). The suppression effect
on tumor proliferation at the inoculated sites were measured in the
same manner as Example 3, i.e. by measuring the size of each
subcutaneous tumor by a caliper every other day, thus the volumes
of tumors were measured (V; mm.sup.3).
[0075] The results of the above are shown in FIG. 12. As a result,
the suppression effect on tumor proliferation was strongly
suppressed in a group wherein splenic T cells derived from rats
immunized with Tax 180-188 and ISS-ODN (*) were transfected,
however, such suppression effect on tumor proliferation was not
recognized in the group transfected with splenic T cells derived
from rats immunized respectively with the following: Tax 180-188
peptide only (.box-solid.); ISS-ODN only (.tangle-solidup.);
Influenza A matrix 58-66 and ISS-ODN (.diamond-solid.). As a result
of the above-mentioned, it is revealed to be possible to induce Tax
180-188-specific CTLs to immunocompetent rats by using an
appropriate adjuvant, and that such induced CTLs can strongly
suppress the proliferation of HTLV-I-infected tumor cells in vivo.
These results show that Tax 180-188 can act independently as a
tumor rejection antigen, and that a vaccine wherein Tax 180-188
peptide and ISS-ODN are used together efficiently induce T cell
immunity and protect from HTLV-I-infected tumors in vivo, and that
the present experimental system is extremely useful as a
developmental model of a vaccine.
[0076] The invention is further described by the following numbered
paragraphs:
[0077] 1. A screening method for a CTL recognition antigen or an
antigen epitope thereof which can induce cytotoxic T lymphocytes
(CTLs) having an anti-tumor effect against HTLV-I tumors, wherein
CTLS induced by a test substance is administered to a non-human
animal model of HTLV-I-associated disease, and the change of tumors
in said non-human animal is measured and assessed.
[0078] 2. The screening method for a CTL recognition antigen or an
antigen epitope thereof which can induce CTLs having an anti-tumor
effect against HTLV-I tumors according to paragraph 1, wherein the
non-human animal model of HTLV-I-associated disease is a non-human
animal model of adult T cell leukemia.
[0079] 3. The screening method for a CTL recognition antigen or an
antigen epitope thereof which can induce CTLs having an anti-tumor
effect against HTLV-I tumors according to paragraph 1 or 2, wherein
the non-human animal is a rat.
[0080] 4. A screening method for a CTL recognition antigen or an
antigen epitope thereof which can induce CTLs having an anti-tumor
effect against HTLV-I tumors, wherein CTLs induced by a test
substance is contacted with HTLV-I-infected tumor cell lines, and
the cytotoxic activity of said CTLs is measured and assessed.
[0081] 5. A screening method for a CTL recognition antigen or an
antigen epitope thereof which can induce CTLs having an anti-tumor
effect against HTLV-I tumors, wherein target cells sensitized with
a test substance or target cells that express a test substance are
contacted with HTLV-I-specific CTL cell lines, and the cytotoxic
activity of said HTLV-I-specific CTL cell lines is measured and
assessed.
[0082] 6. The CTL recognition antigen or the antigen epitope
thereof which can induce CTLs having an anti-tumor effect against
HTLV-I tumors, which is obtained by the screening methods according
to any of paragraphs 1 to 5.
[0083] 7. The antigen epitope according to claim 6, wherein the
antigen epitope is a peptide comprising an amino acid sequence
shown by Seq. ID No. 2.
[0084] 8. A screening method for an adjuvant that enhances an
anti-tumor effect against HTLV-I tumors, wherein CTLs induced by
using the CTL recognition antigen or the antigen epitope thereof
according to paragraph 6 and a test adjuvant are administered to a
non-human animal model of HTLV-I-associated diseases and the change
of tumors in said non-human animals is measured and assessed.
[0085] 9. The screening method for an adjuvant that enhances an
anti-tumor effect against HTLV-I tumors according to paragraph 8,
wherein the non-human animal model of HTLV-I-associated disease is
a non-human animal model of adult T cell leukemia.
[0086] 10. The screening method for an adjuvant that enhances an
anti-tumor effect against HTLV-I tumors according to paragraph 8 or
9, wherein the non-human animal is a rat.
[0087] 11. A screening method for an adjuvant that enhances an
anti-tumor effect against HTLV-I tumors, wherein CTLs induced by
using the CTL recognition antigen or the antigen epitope thereof
according to paragraph 6 and a test adjuvant are contacted with
HTLV-I-infected tumor cell lines, and the cytotoxic activity of
said CTLs is measured and assessed.
[0088] 12. The screening method for an adjuvant that enhances an
anti-tumor effect against HTLV-I tumors according to any of
paragraphs 8 to 11, wherein the antigen epitope peptide according
to paragraph 7 is used as the CTL recognition antigen or the
antigen epitope thereof according to paragraph 6.
[0089] 13. A vaccine for inducing an immune response containing a
CTL recognition antigen or an antigen epitope thereof which can
induce CTLs having an anti-tumor effect against HTLV-I tumors and
which can be obtained by the screening method according to any of
paragraph 1 to 5 as an active ingredient.
[0090] 14. A vaccine for inducing an immune response containing a
DNA that encodes a CTL recognition antigen or an antigen epitope
thereof which can induce CTLs having an anti-tumor effect against
HTLV-I tumors obtained by the screening method according to any of
paragraphs 1 to 5 as an active ingredient.
[0091] 15. The vaccine for inducing an immune response according to
paragraph 13 or 14, wherein the CTL recognition antigen is an
HTLV-I Tax protein shown by Seq. ID No.1.
[0092] 16. The vaccine for inducing an immune response according to
paragraph 13 or 14, wherein the CTL recognition antigen is a
protein comprising an amino acid sequence wherein one or a few
amino acids are deleted, substituted, or added in the amino acid
sequence shown by Seq. ID No.1, which can induce CTLs having an
anti-tumor action against HTLV-I tumors.
[0093] 17. The vaccine for inducing an immune response according to
paragraph 13 or 14, wherein the CTL recognition antigen epitope is
a peptide comprising the amino acid sequence shown by Seq. ID
No.2.
[0094] 18. The vaccine for inducing an immune response according to
paragraph 13 or 14, wherein the CTL recognition antigen epitope is
a peptide comprising an amino acid sequence wherein one or a few
amino acids are deleted, substituted, or added in the amino acid
sequence shown by Seq. ID No. 2, which can induce the CTLs having
an anti-tumor action against HTLV-I tumors.
[0095] 19. The vaccine for inducing an immune response according to
any of paragraphs 13 to 18, further containing an adjuvant that
enhances an anti-tumor effect against HTLV-I tumors.
[0096] 20. The vaccine for inducing an immune response according to
paragraph 19, wherein the adjuvant which can be obtained by the
screening method according to any of paragraphs 8 to 12 is an
adjuvant that enhances an anti-tumor effect against HTLV-I
tumors.
[0097] 21. The vaccine for inducing an immune response according to
paragraph 20, wherein the adjuvant that enhances an anti-tumor
effect against HTLV-I tumors which can be obtained by the screening
method according to any of paragraphs 8 to 12 is ISS-ODN.
[0098] 22. An HTLV-I recognition CTL induced by the vaccine for
inducing an immune response according to any of paragraphs 13 to
21.
[0099] 23. A pharmaceutical composition containing the HTLV-I
recognition CTL according to paragraph 22 as an active
ingredient.
[0100] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the appended claims is not to be limited to particular
details set forth in the above description, as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention. Modifications and variations of
the method and apparatuses described herein will be obvious to
those skilled in the art, and are intended to be encompassed by the
following claims.
Sequence CWU 1
1
6 1 353 PRT Human T-cell lymphotropic virus type 1 1 Met Ala His
Phe Pro Gly Phe Gly Gln Ser Leu Leu Phe Gly Tyr Pro 1 5 10 15 Val
Tyr Val Phe Gly Asp Cys Val Gln Gly Asp Trp Cys Pro Ile Ser 20 25
30 Gly Gly Leu Cys Ser Ala Arg Leu His Arg His Ala Leu Leu Ala Thr
35 40 45 Cys Pro Glu His Gln Ile Thr Trp Asp Pro Ile Asp Gly Arg
Val Ile 50 55 60 Gly Ser Ala Leu Gln Phe Leu Ile Pro Arg Leu Pro
Ser Phe Pro Thr 65 70 75 80 Gln Arg Thr Ser Lys Thr Leu Lys Val Leu
Thr Pro Pro Ile Thr His 85 90 95 Thr Thr Pro Asn Ile Pro Pro Ser
Phe Leu Gln Ala Met Arg Lys Tyr 100 105 110 Ser Pro Phe Arg Asn Gly
Tyr Met Glu Pro Thr Leu Gly Gln His Leu 115 120 125 Pro Thr Leu Ser
Phe Pro Asp Pro Gly Leu Arg Pro Gln Asn Leu Tyr 130 135 140 Thr Leu
Trp Gly Gly Ser Val Val Cys Met Tyr Leu Tyr Gln Leu Ser 145 150 155
160 Pro Pro Ile Thr Trp Pro Leu Leu Pro His Val Ile Phe Cys His Pro
165 170 175 Gly Gln Leu Gly Ala Phe Leu Thr Asn Val Pro Tyr Lys Arg
Ile Glu 180 185 190 Glu Leu Leu Tyr Lys Ile Ser Leu Thr Thr Gly Ala
Leu Ile Ile Leu 195 200 205 Pro Glu Asp Cys Leu Pro Thr Thr Leu Phe
Gln Pro Ala Arg Ala Pro 210 215 220 Val Thr Leu Thr Ala Trp Gln Asn
Gly Leu Leu Pro Phe His Ser Thr 225 230 235 240 Leu Thr Thr Pro Gly
Leu Ile Trp Thr Phe Thr Asp Gly Thr Pro Met 245 250 255 Ile Ser Gly
Pro Cys Pro Lys Asp Gly Gln Pro Ser Leu Val Leu Gln 260 265 270 Ser
Ser Ser Phe Ile Phe His Lys Phe Gln Thr Lys Ala Tyr His Pro 275 280
285 Ser Phe Leu Leu Ser His Gly Leu Ile Gln Tyr Ser Ser Phe His Ser
290 295 300 Leu His Leu Leu Phe Glu Glu Tyr Thr Asn Ile Pro Ile Ser
Leu Leu 305 310 315 320 Phe Asn Glu Lys Glu Ala Asp Asp Asn Asp His
Glu Pro Gln Ile Ser 325 330 335 Pro Gly Gly Leu Glu Pro Pro Ser Glu
Lys His Phe Arg Glu Thr Glu 340 345 350 Val 2 9 PRT Human T-cell
lymphotropic virus type 1 2 Gly Ala Phe Leu Thr Asn Val Pro Tyr 1 5
3 9 PRT Human T-cell lymphotropic virus type 1 3 Ala Phe Leu Thr
Asn Val Pro Tyr Lys 1 5 4 9 PRT Human T-cell lymphotropic virus
type 1 4 Phe Leu Thr Asn Val Pro Tyr Lys Arg 1 5 5 22 DNA
Artificial Sequence Description of Artificial Sequence adjuvant
ISS-ODN 5 tgactgtgaa cgttcgagat ga 22 6 9 PRT Influenza A virus 6
Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5
* * * * *